Patent Grant
11807881
In the present description, a novel polypeptide, a fusion polypeptide comprising the polypeptide and a use as an antibiotic thereof are provided. More specifically, a novel polypeptide derived from a bacteriophage, a fusion polypeptide comprising the polypeptide and Cecropin A, and an antibiotic against gram negative bacterium comprising the polypeptide and/or fusion protein.
Bacteriophage refers to a bacterium-specific virus that infects specific bacterium and inhibits and hinders the growth of the infected bacterium. The bacteriophage has the ability to kill bacterium in the way of proliferating inside bacterial cells after infection to their host bacterium, and destroy the cell wall of the host bacterium using endolysin, a protein of bacteriophage when progeny bacteriophages come out of the bacterium after proliferation. Therefore, a substance having endolysin activity of a bacteriophage can be usefully applied as an antibiotic candidate.
Recently, antibiotic-resistant bacterium is rapidly increasing, and multidrug-resistant bacterium that cannot be treated with any antibiotic are also increasing. In particular, Pseudomonas aeruginosa, one of gram negative bacteria, is one of ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) bacteria that urgently need development of new treatment methods worldwide, and E. coli (Escherichia coli) is a bacterium involved in various infections. Therefore, it is required to develop a treatment method that is differentiated from the conventional antibiotics.
One example provides a novel polypeptide. The polypeptide may comprise the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 6. The polypeptide may be derived from a bacteriophage or a variant thereof. The polypeptide may have endolysin activity.
Another example provides a polynucleotide encoding the polypeptide. The polynucleotide may comprise the nucleic acid sequence of SEQ ID NO: 2 or SEQ ID NO: 7.
Other example provides a recombinant vector comprising the polynucleotide. The recombinant vector may be used as an expression vector.
Other example provides a recombinant cell comprising the polynucleotide or a recombinant vector comprising the same. The recombinant cell may be for expression of the polynucleotide.
Other example provides a bacteriophage comprising the polypeptide of SEQ ID NO: 1, a polynucleotide encoding the same (for example, SEQ ID NO: 2), or a combination thereof. The genome of the bacteriophage may comprise the nucleic acid sequence of SEQ ID NO: 5.
Other example provides a novel fusion polypeptide. The fusion polypeptide may comprise Cecropin A and endolysin. The Cecropin A may be represented, for example, by the amino acid sequence of SEQ ID NO: 8. The endolysin may be derived from a bacteriophage or a variant thereof, and for example, it may be represented by the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 6. In the fusion polypeptide, the Cecropin A (e.g., SEQ ID NO: 8) and endolysin (for example, SEQ ID NO: 1 or SEQ ID NO: 6) may be linked through a linker in any order or directly linked without a linker. In one example, the fusion polypeptide may be represented by the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 13.
Other example provides a polynucleotide encoding the fusion polypeptide. The polynucleotide may comprise the nucleic acid sequence of SEQ ID NO: 11 or SEQ ID NO: 14.
Other example provides a recombinant vector comprising the polynucleotide. The recombinant vector may be used as an expression vector.
Other example provides a recombinant cell comprising the polynucleotide or a recombinant vector comprising the same. The recombinant cell may be for expression of the polynucleotide.
Other example,
The antibiotic may have antibiotic effect against gram negative bacterium.
The antibiotic (the first antibiotic) provide in the present description has an advantage to exhibit a synergy effect by the antibiotic effect itself and an excellent antibiotic effect as the second antibiotic at a low concentration, as used in combination with another antibiotic (the second antibiotic). For this reason, side effects such as toxicity (for example, kidney toxicity, liver toxicity, etc.) by use of an antibiotic at a high concentration may be reduced. In addition, it can inhibit emergence of antibiotic-resistant bacterium through combination of antibiotics of different mechanisms.
Accordingly, one example,
Other example provides a pharmaceutical composition for preventing and/or treating infection of Pseudomonas sp. bacterium or disease caused by Pseudomonas sp. bacterium comprising the antibiotic or combination antibiotic as an active ingredient. Other example provides a method for preventing or treating infection of Pseudomonas sp. bacterium or disease caused by Pseudomonas sp. bacterium, comprising administering a pharmaceutically effective dose of the antibiotic or combination antibiotic into a subject in need of preventing and/or treating infection of Pseudomonas sp. bacterium or disease caused by Pseudomonas sp. bacterium.
Other example provides a feed additive comprising the antibiotic or combination antibiotic as an active ingredient.
Other example provides a disinfectant comprising the antibiotic or combination antibiotic as an active ingredient.
Other example provides a method for disinfection, comprising applying the antibiotic or combination antibiotic into a subject in need of disinfection.
Other example provides a detergent comprising the antibiotic or combination antibiotic as an active ingredient.
Other example provides a method for cleaning, comprising applying the antibiotic or combination antibiotic into a subject in need of cleaning.
When a bacteriophage that can be used as a natural antibiotic by proliferating bacterium as a host penetrates the bacterium and finishes proliferation inside, the completed phage particles are released to the outside of the bacterium, and then, an enzyme that attacks and decomposes the cell wall of bacterium and creates a pathway for release to the outside is endolysin. All bacteriophages have this endolysin gene in their genome and use the endolysin protein expressed during proliferation. Bacteriophages that proliferate bacterium as a host and endolysin derived from bacteriophages having a property of decomposing the cell wall can be used as natural antibiotics.
In case of gram positive bacterium, as the cell wall is located on the outermost wall, when endolysin is added from the outside, the cell wall is immediately attacked and decomposed. On the other hand, in case of gram negative bacterium, the outer cell membrane is present at the outermost part and the cell wall is located inside it, and therefore, even if endolysin is added from the outside, it must first pass through the outer cell membrane to meet the cell wall. Thus, it has been known that basically endolysin has no effect on gram negative bacterium. The polypeptide having endolysin activity provided in the present description is characterized by having an effect of killing gram negative bacterium.
On the other hand, the Cecropin A is a peptide having antibacterial activity which has action of lysing the bacterial membrane.
In the present description, a novel polypeptide or a fusion polypeptide in which Cecropin A is fused to the novel polypeptide demonstrates a superior antibiotic effect than conventional endolysin, and thereby, a polypeptide having endolysin activity, and an antibiotic thereof and/or a use related thereto are provided.
Hereinafter, the present invention will be described in more detail:
In the present description, that a polynucleotide (may be used interchangeably with “gene”) or a polypeptide (may be used interchangeably with “protein” “comprises a specific nucleic acid sequence or amino acid sequence” or “consists of a specific nucleic acid sequence or amino acid sequence” may mean that the polynucleotide or polypeptide essentially includes the specific nucleic acid sequence or amino acids sequence, and it may be interpreted as including a “substantially equivalent sequence” in which a mutation (deletion, substitution, modification and/or addition) is added to the specific nucleic acid sequence or amino acid sequence within a range of maintaining the original function and/or desired function of the polynucleotide or polypeptide (or not excluding the mutation).
In one example, that a polynucleotide or polypeptide “comprises a specific nucleic acid sequence or amino acid sequence” or “consists of or is represented by a specific nucleic acid sequence or amino acid sequence” may mean that the polynucleotide or polypeptide (i) essentially comprises the specific nucleic acid sequence or amino acid sequence, or (ii) consists of or an amino acid sequence having identity of 96% or more, 96.5% or more, 97% or more, 97.5% or more, 98% or more, 98.5% or more, 99% or more, 99.5% or more, or 99.9% or more to the specific nucleic acid sequence or amino acid sequence or essentially comprises that, and maintains the original function and/or desired function. In the present description, the original function may be endolysin enzymatic function (for example, peptidoglycan hydrolytic activity), Cecropin A activity (for example, cell membrane lytic activity), and/or antibiotic action (in case of amino acid sequence), or endolysin enzymatic function, or function encoding protein having Cecropin A activity and/or antibiotic action (in case of nucleic acid sequence), and the desired function may mean antibiotic activity against gram negative bacterium, for example, Pseudomonas sp. bacterium, for example, Pseudomonas aeruginosa.
In the present description, the term, “identity” means a degree of correspondence with a given nucleic acid sequence or amino acid sequence and may be represented by a percentage (%). In case of identity for a nucleic acid sequence, for example, it may be determined by using algorithm BLAST by a document (reference: Karlin and Altschul, Pro. Natl. Acad. Sci. USA, 90, 5873, 1993) or FASTA by Pearson (reference: Methods Enzymol., 183, 63, 1990). Based on this algorithm BLAST, programs called BLASTN or BLASTX have been developed.
The protein or polypeptide provided in the present description may be separated and/or purified from the nature, or be recombinantly or chemically synthesized. When the amino acid sequence of the protein or polypeptide provided in the present description comprises a methionine (Met, M) or Met-Ala-Ser (MAS) sequence as the first amino acid residue from the N-terminus, the protein or polypeptide may be recombinantly produced, and the methionine at the first amino acid position from the N-terminus may be encoded by an initiation codon. Therefore, when the amino acid sequence of the protein or polypeptide provided in the present description comprises methionine by recombinant production at the N-terminus, and when the protein or polypeptide is obtained by other method (for example, chemical synthesis or separation from the nature), it may be interpreted as comprising an amino acid sequence starting from the second amino acid residue excluding the methionine at the first position from the N-terminus by the recombinant production or an amino acid sequence starting from the amino acid residue following the MAS sequence (for example, the 4th amino acid residue).
One example provides a novel polypeptide. The polypeptide may comprise the amino acid sequence of SEQ ID NO: 1. The polypeptide may be derived from a bacteriophage. The polypeptide may have endolysin activity derived from a bacteriophage. The polypeptide may have a molecular weight of about 28 kDa (28 kDa±2).
In another example, the polypeptide may comprise the amino acid sequence of SEQ ID NO: 6. The polypeptide may be an endolysin variant derived from a bacteriophage, and may have excellent endolysin activity and/or antibiotic activity compared to endolysin derived from a bacteriophage. The polypeptide may have a molecular weight of about 28 kDa (28 kDa±2). The polypeptide may be prepared recombinantly or synthetically (for example, chemical synthesis).
Other example provides a polynucleotide encoding the polypeptide. The polynucleotide may comprise the nucleic acid sequence of SEQ ID NO: 2 or SEQ ID NO: 7.
Endolysin refers to a peptidoglycan hydrolase encoded by a bacteriophage. Endolysin is synthesized during late gene expression in the lytic cycle of bacteriophage proliferation and mediates the release of progeny virions from infected cells through degradation of bacterial peptidoglycan. In respect of enzymatic activity, endolysin may commonly have one or more activities selected from the group consisting of glucosaminidase, muramidase (one kind of lysozyme), transglycosylase, amidase, endopeptidase, and the like.
In the present description, unless otherwise described, the polypeptide having endolysin activity as a polypeptide ‘comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 6’ or ‘consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 6’,
In addition, in the present description, unless otherwise described, a polynucleotide encoding a polypeptide ‘comprising the amino acid sequence of ‘SEQ ID NO: 1 or SEQ ID NO: 6’ or ‘consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 6’,
Other example provides a novel fusion polypeptide. The fusion polypeptide may comprise Cecropin A and endolysin.
The Cecropin A may be derived from a moth (Hyalophora cecropia), and for example, may be represented by the amino acid sequence of SEQ ID NO: 8. The endolysin may be derived from a bacteriophage or a variant thereof, and for example, it may be represented by the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 6.
In the fusion polypeptide, the Cecropin A (for example, SEQ ID NO: 8) and endolysin (for example, SEQ ID NO: 1 or SEQ ID NO: 6) may be linked in any order. In other words, in the fusion polypeptide, the Cecropin A and endolysin may be linked in the order of Cecropin A and endolysin or the order of endolysin and Cecropin A, and for example, it may be linked in the order of Cecropin A and endolysin.
In addition, the Cecropin A (for example, SEQ ID NO: 8) and endolysin (for example, SEQ ID NO: 1 or SEQ ID NO: 6) may be linked through a linker or directly linked without a linker.
In one example, the fusion polypeptide may be represented by the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 13.
The fusion polypeptide may have excellent antibiotic activity and/or excellent outer membrane permeabilization against gram negative bacterium, compared to endolysin or Cecropin A. The fusion polypeptide may have a molecular weight of about 34 kDa (for example, 34 kDa±3). The fusion polypeptide or endolysin and Cecropin A may be prepared recombinantly or synthetically (for example, chemical synthesis).
Other example provides a polynucleotide encoding the fusion polypeptide. The polynucleotide may comprise the nucleic acid sequence of SEQ ID NO: 11 or SEQ ID NO: 14.
The novel polypeptide provided in the present description (for example, SEQ ID NO: 1 or SEQ ID NO: 6) has endolysin activity. Endolysin means peptidoglycan hydrolase encoding a bacteriophage. Endolysin is synthesized during later gene expression in a lytic cycle of bacteriophage proliferation and mediates the release of progeny virions from infected cells through degradation of bacterial peptidoglycan. In respect of enzymatic activity, endolysin may commonly have one or more activities selected from the group consisting of glucosaminidase, muramidase (one kind of lysozyme), transglycosylase, amidase, endopeptidase, and the like.
In the present description, unless otherwise described, the fusion polypeptide,
In one example, the fusion polypeptide,
In addition, in the present description, unless otherwise described, a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 10,
In the fusion polypeptide provided in the present description, Cecropin A and endolysin may be linked through a peptide linker or directly linked without a peptide linker. The peptide linker may be a polypeptide consisting of any amino acids of 1 to 100, 2 to 50, 1 to 30, 2 to 20 or 2 to 10, and the kind of the amino acids comprised therein has no limitation. The peptide linker may comprise one or more kinds of amino acid residues selected from the group consisting of for example, Gly, Ser, Leu, Gln, Asn, Thr and Ala, respectively. The amino acid sequence suitable for the peptide linker is known in the art. On the other hand, the length of the linker may be variously determined within a limit that does not affect the structure and/or function of the fusion protein. For example, the peptide linker may be composed as including a total of 1 to 100, 2 to 50, 1 to 30, 2 to 20 or 2 to 10 of one or more kinds selected from the group consisting of Gly, Ser, Leu, Gln, Asn, Thr and Ala. In one example, the peptide linker may be represented by GSGSGS (SEQ ID NO: 12).
Recombinant Vector, and Recombinant Cell
Other example provides a recombinant vector comprising the aforementioned polypeptide (a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 6) or a polynucleotide encoding the aforementioned fusion polypeptide (for example, SEQ ID NO: 10 or SEQ ID NO: 13). The recombinant vector may be used as an expression vector.
Other example provides a method for preparation of the polypeptide or the fusion polypeptide, comprising expression the polypeptide or fusion polypeptide in an appropriate host cell. The expression the polypeptide or the fusion polypeptide in an appropriate host cell may be performed by culturing the polynucleotide or recombinant vector comprising the same in a recombinant cell. The method for preparation of endolysin, may further comprise separating and/or purifying expressed endolysin, after the expression.
Introduction of the polynucleotide or vector into a host cell may be performed by appropriately selecting a transformation method. In the present description, the term “transformation” refers to introducing a vector comprising a polynucleotide encoding the polypeptide into a host cell so that the polypeptide encoding the polynucleotide can be expressed. The transformed polynucleotide may include all, whether inserted and located in the chromosome of the host cell or located extrachromosomally, as long as they can be expressed in the host cell. The type of the polynucleotide to be introduced is not limited, as long as it is introduced into the host cell and expressed. For example, the polynucleotide may be introduced into a host cell in a form of expression cassette which is a gene structure comprising all elements required for self-expression. The expression cassette may commonly comprise expression regulatory elements such as a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding site and/or a translation termination signal, and the like. The expression cassette may be in a form of expression vector capable of self replication. In addition, the polynucleotide may be introduced into a host cell in its own form and be operably linked to a sequence required for expression in a host cell. In the above, the term “operably linked” may mean that an expression regulatory element (e.g., promoter) and a polynucleotide are functionally linked to perform transcriptional regulation (e.g., transcription initiation) of the polynucleotide. Operable linking may be performed using a genetic recombinant technique known in the art.
The method for transforming the polynucleotide into a host cell may be performed by any method for introducing a nucleic acid into a cell (microorganism), and may be performed by appropriately selecting a transformation technique known in the art depending on the host cell. As the known transformation method, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) precipitation (polyethylene glycol-mediated uptake), DEAE-dextran method, cation liposome method, lipofection, lithium acetate-DMSO method, and the like may be exemplified, but not limited thereto.
In the present description, the term “vector” refers to a DNA structure containing a nucleotide sequence of a polynucleotide operably linked to an appropriate regulatory sequence so as to express a target protein in a suitable host. The regulatory sequence may comprise a promoter capable of initiating transcription, any operator sequence for regulating transcription, a sequence encoding an appropriate mRNA ribosome binding site, and/or a sequence regulating termination of transcription and/or translation. The vector may be expressed regardless of genome of a host cell or interpreted in the genome of the host cell, after transformed into a proper host cell.
The vector available in the present description is not particularly limited as long as it can be replicated in a host cell, and may be selected from all commonly used vectors. The example of the commonly used vectors may include a natural or recombinant plasmid, cosmid, virus, bacteriophage, or the like. For example, as the vector, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A and the like may be used as the phage vector or cosmid vector, and pBR-based, pUC-based, pBluescriptII-based, pGEM-based, pTZ-based, pCL-based and pET-based, and the like may be used as the plasmid vector. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, and the like may be exemplified, but not limited thereto.
The vector may further comprise a selection marker for confirming whether inserted in the chromosome or not. The selection marker is for selecting transformed cells with a vector, that is, confirming insertion of the polynucleotide, and genes that confer selectable phenotypes such as drug resistance, auxotrophy, resistance to cytotoxic agents or expression of surface proteins may be selected and used. In an environment treated with a selective agent, only cells expressing a selection marker survive or exhibit other expression traits and therefore, transformed cells may be selected.
The polypeptide provided in the present description may be not naturally derived, and may be synthesized recombinantly or chemically. When the polypeptide is recombinantly produced, it may be in a form in which a common signal peptide, a cleavage site, a tag, and the like are combined for purification. Therefore, in one non-limitative example, the polypeptide provided in the present description may be in a form further comprising one or more selected from the group consisting of a signal peptide, a cleavage site, a tag (for example, His tag, GST (glutathione-s-transferase) tag, MBP (maltose binding protein) tag, etc.), and the like, which can be commonly used in the process of recombinant production of a protein, or may be in a purified form in which they are removed.
Other example,
The antibiotic may have an antibiotic effect against gram negative bacterium. The gram negative bacterium may be one or more kinds (for example, 1 kind, 2 kinds, 3 kinds, 4 kinds or 5 kinds) selected from the group consisting of Pseudomonas sp. bacterium, Acinetobacter sp. bacterium, Escherichia sp. bacterium, Enterobacter sp. bacterium, Klebsiella sp. bacterium, and the like. For example, the Pseudomonas sp. bacterium may be Pseudomonas aeruginosa, and the Acinetobacter sp. bacterium may be Acinetobacter baumannii, and the Escherichia sp. bacterium may be Escherichia coli, and the Enterobacter sp. bacterium may be Enterobacter aerogenes, and the Klebsiella sp. bacterium may be Klebsiella pneumoniae, but not limited thereto.
The antibiotic provided in the present description may further comprise another antibiotic (the second antibiotic), in addition to one or more kinds selected from the group consisting of the aforementioned polypeptide (polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 6) or the fusion polypeptide, a polynucleotide encoding the polypeptide or the fusion polypeptide, a recombinant vector comprising the polynucleotide, and a recombinant cell comprising the polynucleotide or a recombinant vector comprising the same (hereinafter, the first antibiotic).
The second antibiotic may be one or more kinds selected from commonly used antibiotics, for example, antibiotics having antibiotic activity against gram negative bacterium. In one specific example, the second antibiotic may be a polymyxin-based antibiotic, and for example, it may be polymyxin B, Colistin, or a combination thereof, but not limited thereto.
As such, by using the antibiotic (the first antibiotic) with other antibiotic (the second antibiotic) in combination, it has an advantage of exhibiting a synergy effect by the antibiotic effect of the first antibiotic itself and an excellent antibiotic effect as the second antibiotic at a low concentration. Due to this, side effects such as toxicity (for example, kidney toxicity, liver toxicity, etc.) by use of an antibiotic at a high concentration may be reduced. In addition, emergence of antibiotic-resistant bacterium through use in combination of an antibiotic of other mechanism may be inhibited.
Accordingly, one example,
In the present description, the term “antibiotic” encompasses all the types of agents having growth inhibitory ability and/or killing ability against gram negative bacterium, and unless otherwise mentioned, it may be interchangeably used with antibacterial agent, preservative, disinfectant, or the like.
Other example provides a pharmaceutical composition for preventing and/or treating infection of gram negative bacterium or disease caused by gram negative bacterium comprising the antibiotic or combination antibiotic.
Other example provides a method for preventing or treating infection of gram negative bacterium or disease caused by gram negative bacterium, administering a pharmaceutically effective dose of the antibiotic or combination antibiotic into a subject in need of preventing and/or treating infection of gram negative bacterium or disease caused by gram negative bacterium. The method for preventing or treating, may further comprise confirming a subject in need of preventing and/or treating infection of gram negative bacterium or disease caused by gram negative bacterium, before the administering. The gram negative bacterium is same as described above.
The disease caused by gram negative bacterium may be selected from all diseases caused by infection of gram negative bacterium, and for example, it may be selected from the group consisting of disease caused by Pseudomonas sp. bacterium such as skin infection, bedsore, pneumonia, bacteremia, septicemia, endocarditis, meningitis, otitis externa, otitis media, keratitis, osteomyelitis, enteritis, peritonitis or cystic fibrosis; disease caused by Acinetobacter sp. bacterium such as skin infection, pneumonia, bacteremia or septicemia; disease caused by Escherichia sp. bacterium such as enteritis, Crohn's disease, ulcerative colitis, bacillary dysentery, urinary tract infection, skin infection, bacteremia or septicemia, and the like, but not limited thereto.
As used in the present description, the pharmaceutically effective dose refers to a contained amount or dosage of an active ingredient capable of obtaining a desired effect. The contained amount or dosage of an active ingredient in the pharmaceutical composition may be variously prescribed by factors such as preparation method, administration method, patient's age, body weight, gender, morbidity, food, administration time, administration interval, administration route, excretion rate and reaction sensitivity. For example, when the active ingredient is a polypeptide, a single dose may be in a range of 0.001 to 1000 mg/kg, 0.01 to 100 mg/kg, 0.01 to 50 mg/kg, 0.01 to 20 mg/kg, 0.01 to 10 mg/kg, 0.01 to 5 mg/kg, 0.1 to 100 mg/kg, 0.1 to 50 mg/kg, 0.1 to 20 mg/kg, 0.1 to 10 mg/kg, 0.1 to 5 mg/kg, 1 to 100 mg/kg, 1 to 50 mg/kg, 1 to 20 mg/kg, 1 to 10 mg/kg, or 1 to 5 mg/kg, but not limited thereto.
In other example, the content of the active ingredient in the pharmaceutical composition may be 0.01% by weight to 99.9% by weight, 0.01% by weight to 90% by weight, 0.01% by weight to 80% by weight, 0.01% by weight to 70% by weight, 0.01% by weight to 60% by weight, 0.01% by weight to 50% by weight, 0.01% by weight to 40% by weight, 0.01% by weight to 30% by weight, 1% by weight to 99.9% by weight, 1% by weight to 90% by weight, 1% by weight to 80% by weight, 1% by weight to 70% by weight, 1% by weight to 60% by weight, 1% by weight to 50% by weight, 1% by weight to 40% by weight, 1% by weight to 30% by weight, 5% by weight to 99.9% by weight, 5% by weight to 90% by weight, 5% by weight to 80% by weight, 5% by weight to 70% by weight, 5% by weight to 60% by weight, 5% by weight to 50% by weight, 5% by weight to 40% by weight, 5% by weight to 30% by weight, 10% by weight to 99.9% by weight, 10% by weight to 90% by weight, 10% by weight to 80% by weight, 10% by weight to 70% by weight, 10% by weight to 60% by weight, 10% by weight to 50% by weight, 10% by weight to 40% by weight, or 10% by weight to 30% by weight, based on the total weight of the pharmaceutical composition, but not limited thereto.
In addition, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, in addition to the active ingredient. The pharmaceutically acceptable carrier may refer to a carrier which is commonly used in preparation of a drug comprising a protein, nucleic acid or cell, and does not stimulate an organism and does not inhibit the biological activity and/or properties of an active ingredient. In one example, the carrier may be one or more kinds selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like, but not limited thereto. The pharmaceutical composition may further comprise one or more kinds selected from the group consisting of a diluent, an excipient, a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifying agent, a suspending agent, a preservative and the like, which are commonly used in preparation of a pharmaceutical composition.
The administration subject of the pharmaceutical composition may be one or more kinds selected from a mammal including primates such as human and monkeys, rodents such as mice and rats, livestock such as dogs, cats, pigs, cattle, horses, sheep and goats, poultry such as chickens, ducks, geese, pheasants, quails and turkeys, and the like, or a cell, tissue or culture thereof derived therefrom.
The pharmaceutical composition may be administered by oral administration or parenteral administration, or be administered by contacting to a cell, tissue or body fluid. Specifically, in case of parenteral administration, it may be administered by subcutaneous injection, intramuscular injection, intravenous injection, intraperitoneal injection, endothelial administration, local administration, intranasal administration, intrapulmonary administration and intrarectal administration, and the like. In case of oral administration, as a protein or peptide is digested, the oral composition should be formulated to coat an active agent or to protect it from degradation in stomach. In case of intranasal administration, the pharmaceutical composition may be administered through nasal spray to dilute it and to be absorbed into nasal cavity through a sprayer or spray system, and a nasal sprayer or a respiratory formulation for the nasal spray may include an aerosol.
In addition, the pharmaceutical composition may be formulated in a form of solution in an oil or aqueous medium, injection, suspension, syrup, emulsion, ointment, patch, extract, powder, powder, granule, tablet, capsule, aerosol, or the like, and it may further comprise a dispersant or a stabilizer for formulation.
Other example provides a feed additive comprising the antibiotic or combination antibiotic as an active ingredient.
Other example provides a feed comprising the composition for the feed additive.
For the feed, the antibiotic or combination antibiotic may be prepared separately in a form of feed additive and mixed to the feed, or it may be prepared by directly adding it during feed preparation.
The antibiotic or combination antibiotic in the feed may be in liquid or dried form, and for example, it may be a dried powder form. The antibiotic may be comprised in an amount of 0.005 to 10% by weight, 0.05 to 10% by weight, 0.1 to 10% by weight, 0.005 to 5% by weight, 0.05 to 5% by weight, 0.1 to 5% by weight, 0.005 to 2% by weight, 0.05 to 2% by weight, or 0.1 to 2% by weight of the total feed weight, but not limited thereto. In addition, the feed may further comprise common additives which can increase preservability of the feed in addition to the antibiotic or combination antibiotic.
In the present description, the feed in which the antibiotic or combination antibiotic may be selected from the group consisting of commercially available feed, or grains, root fruits, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, gourds, grain by-products, proteins, inorganic matters, oils and fats, minerals, single cell proteins, zooplankton, leftover food, and the like, but not limited thereto.
Other example provides a food additive or drinking water additive comprising the antibiotic or combination antibiotic as an active ingredient. By mixing the antibiotic or combination antibiotic in drinking water and providing it, the number of gram negative bacterium in drinking water may be reduced. The gram negative bacterium is same as described above.
Other example provides a disinfectant comprising the antibiotic or combination antibiotic as an active ingredient. Other example provides a method for disinfection, comprising applying the antibiotic or combination antibiotic to a subject in need of disinfection. The disinfectant is a generic term for agents to prevent pathogen infection, and may be used for disinfection of general life disinfectants, disinfectants for food and cooking places and facilities, and various kinds of growth and development supplies such as buildings such as poultry farm and pen, livestock bodies, drinking water, litter, egg tray, transport vehicles, tableware, and the like.
Other example provides a detergent comprising the antibiotic or combination antibiotic as an active ingredient. Other example provides a method for cleaning, comprising applying the antibiotic or combination antibiotic into a subject in need of cleaning. As the antibiotic has the antibiotic effect against gram negative bacterium, it may be applied for cleaning (washing) the skin surface or body parts of individuals exposed or likely to be exposed to gram negative bacterium. The gram negative bacterium is same as described above.
The novel polypeptide, variant of the polypeptide or novel fusion polypeptide provided in the present description can be usefully used as an antibiotic against gram negative bacterium, by exhibiting excellent outer membrane permeabilization and killing activity against various gram negative bacterium.
The novel endolysin and/or bacteriophage expressing the same provided in the present description exhibit excellent growth inhibitory ability and killing ability against Pseudomonas sp. bacterium, for example, Pseudomonas aeruginosa, and thereby, they can be usefully used as an antibiotic against Pseudomonas sp. bacterium, for example, Pseudomonas aeruginosa.
Pseudomonas aeruginosa (PR01957) was used as a host, and cultured with shaking in a LB (Luria-Bertani) medium under the condition of 37° C.
In order to select a bacteriophage infected by Pseudomonas aeruginosa, samples were collected from Gwacheon sewage treatment plant, Gwacheon-si, Gyeonggi-do, Korea. The collected samples and Pseudomonas aeruginosa were cultured at 37° C. for 3 hours, and then centrifuged at 500 rpm for 20 minutes and the supernatant was collected. Subsequently, the supernatant was filtered with a 0.45 μm filter, and then double agar layer plaque assay was performed.
Briefly describing the analysis method, the culture solution of the host bacterium, Pseudomonas aeruginosa and bacteriophage was mixed by 0.1 M.O.I. to the top agar 5 ml, and poured to an agar plate, and cultured at 37° C. for 24 hours to obtain a plaque. Through repeated performance of the process, the purified pure bacteriophage was obtained, and this bacteriophage was named bacteriophage PBPA90.
Sequencing for genome of the bacteriophage PBPA90 obtained in the Example 1.1 was conducted. After culturing Pseudomonas aeruginosa in a LB medium of 200 ml to OD600=0.5, here, it was lysed by infection with the filtered bacteriophage 109 pfu/ml or 0.1 M.O.I. After that, sodium chloride was added so that the final concentration was 1 M here, and then it was left at 4° C. for 1 hour. Subsequently, after centrifuging at 11,000× g for 10 minutes, PEG (Polyethylene glycol 8000) was added to precipitates by 10% (w/v), and it was left at 4° C. for 1 hour. In succession, after centrifuging at 11,000× g for 10 minutes, the supernatant was removed, and the precipitates were suspended with SM buffer solution [100 mM NaCl, 10 mM MgSO4 (heptahydrate), 50 mM Tris-HCl, pH 7.5]. Chloroform was added at a ratio of 1:1 here, and voltexing was conducted, and then it was centrifuged at 3,000× g for 15 minutes to obtain the supernatant.
40% (w/v) glycerol 3 ml was added to a polycarbonate test tube, and then, 5% (w/v) glycerol 4 ml was added so as not to be mixed. Subsequently, the supernatant was removed, and then precipitates were resuspended with SM buffer solution to obtain bacteriophage genome DNA. The bacteriophage genome DNA was separated using a phage DNA isolation kit (Norgen biotek corp.) according to the manufacturer's manual. The sequence for genome was analyzed using the genome sample separated as above (LAS, Illumina MiSeq platform).
The finally analyzed bacteriophage PBPA90 genome had the total nucleic acid sequence length of 304,052 bp, and had a 44% GC content. The full-length nucleic acid sequence of the bacteriophage PBPA90 genome was represented in SEQ ID NO: 5. The similarity with the conventionally known bacteriophage genome sequence was investigated using BLAST on Web based on the genome nucleic acid sequence information. As the result of BLAST investigation, it was confirmed that the genome sequence of the bacteriophage PBPA90 had low sequence similarity to Pseudomonas bacteriophage KTN4 (GenBank accession No.: KU521356.1) (query coverage: 4%, identity: 97.34%). Based on this fact, it was confirmed that the bacteriophage PBPA90 is a new bacteriophage which is not conventionally known.
Through ORF search for the genomic sequence of the analyzed bacteriophage PBPA90 (SEQ ID NO: 5), the ORF of 780 bp at positions 180,029-180,806 (SEQ ID NO: 2) was estimated to be an endolysin gene, and the bacteriophage PBPA90-derived endolysin and a gene encoding this were named LNT101 endolysin and LNT101 gene, respectively.
Using Primer (F: 5′-aaggatccatgggtactgtactcaaacgtggc-3′ (SEQ ID NO: 3), R: 5′-aactcgagtgcccgatgtttcgaaactttatcttc-3′ (SEQ ID NO: 4)), for the genome of the bacteriophage PBPA90, PCR (polymerase chain reaction) was performed to obtain LNT101 gene (nucleic acid sequence with a 780 bp length at positions 180,029-180,806 in SEQ ID NO: 5). The amino acid sequence of LNT101 endolysin encoded by the LNT101 gene was represented in SEQ ID NO: 1 (259 aa). The PCR was performed under the following condition: step 1: 94° C., 5 minutes; step 2: 94° C., 30 seconds; step 3: 52° C., 45 seconds; step 4: 72° C., 1 minute; step 5: repeating steps 2-4 30 times; step 6: 72° C., 10 minutes. The obtained PCT products were cloned with BamHI/XhoI site of pET-21a vector (Novagen) having N-terminal 6× His-tag, to prepare an expression vector for LNT101 endolysin expression (pET-LNT101 plasmid). The prepared expression vector pET-LNT101 was schematically shown in
The prepared pET-LNT101 plasmid was transformed in E. coli BL21-pLysS strain (Novagen), and then was cultured in LB broth (1% Tryptone, 0.5% (w/v) Yeast extract, 0.5% (w/v) NaCl) to OD600=0.5. After that, 1 mM IPTG (Isopropyl β-d-1-thiogalactopyranosid) was added, and then was cultured with shaking at 37° C. for 4 hours. After cell harvest, it was resuspended with lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole), and 1 mM PMSF and 1 mg/ml lysozyme were added, and it was left on ice for 30 minutes. Cells were lysed by sonication, and centrifuged at 13,000 rpm for 40 minutes to obtain the supernatant. This was passed through a column in which Ni-NTA agarose resin (Qiagen) was packed. After that, it was washed with wash buffer (50 mM NaH2PO4, 300 mM NaCl, 30 mM imidazole), and then it was eluted with elution buffer (50 mM NaH2PO4, 300 mM NaCl, 300 mM imidazole), to purify LNT101 protein (including 6× His tag).
The purity of the LNT101 protein was confirmed by 15% SDS-PAGE and the concentration of the LNT101 protein was measured by Bradford assay. The result of confirming each reactant obtained in the purification process by SDS-PAGE was shown in
In the present example, in order to investigate a target bacteria spectrum of the bacteriophage PBPA90 selected in the Examples 1.1 and 1.2 and endolysin LNT101 separated and purifying in the Example 1.3, the antibacterial activity against gram negative bacterium such as Pseudomonas aeruginosa ATCC13388, ATCC9027, ATCC10145, ATCC15692, ATCC15522, ATCC25619, ATCC27853, CCARM2134, CCARM2200, CCARM2029, CCARM2144, CCARM2298, CCARM2326, PR01957, E. coli ATCC8739, Enterobacter cloacae CCARM0252, Klebsiella pneumoniae KCTC2261, Klebsiella aerogenes CCARM16006, Campylobacter jejuni KCTC5327, Cronobacter sakazakii KCTC2949, Salmonella typhimurium ATCC14028, Salmonella enteritidis ATCC13076 was tested.
The target bacteria spectrum of the bacteriophage PBPA90 and endolysin LNT101 was confirmed by a spot test. For the spot test, bacterium sterilized in 4 ml top agar (1% Tryptone, 0.5% Yeast extract, 0.5% NaCl, 0.7% agar) of 1×1011 CFU/200 μl were added and poured to a LB plate. After hardening the top agar, the bacteriophage PBPA90 10 μl (1×108 PFU/ml) or LNT101 10 μl (2 mg/ml) was spotted, and it was incubated at 37° C. for 18 hours.
Production of the halo zone formed as the result of the incubation was confirmed, and the result was shown in Table 1 below:
P. aeruginosa
E. coli
E. cloacae
K. pneumoniae
K. aerugenes
C. jejuni
C. sakazakii
S. typhimurium
S. enteritidis
As shown in Table 1, the endolysin LNT101 formed the halo zone in all the tested gram negative bacterium, which were Pseudomonas aeruginosa, E, coli, Enterobacter cloacae, Klebsiella pneumoniae, Campylobacter, Cronobacter, and Salmonella strains. This result shows that the endolysin LNT101 has the degradation ability for peptidoglycans of various gram negative bacterium as aforementioned and has a broad target bacterium spectrum.
In the present example, for Pseudomonas aeruginosa (ATCC 13388) confirmed against which the bacteriophage PBPA90 and endolysin LNT101 had the antibacterial activity in the Example 1.4, the bacterium killing ability of endolysin LNT101 was tested.
For this, the endolysin LNT101 at a concentration of 0.1, 0.5, 1.0 μM and Pseudomonas aeruginosa (ATCC 13388) 1Δ106 CFU were added so that the final volume was 200 μl to reaction buffer (20 mM Tris-Cl, pH 7.5) and they were left at 37° C. for 1 hour. In 30 minutes, 1 hour and 2 hours, the number of colonies of Pseudomonas aeruginosa was confirmed, and the result was shown in
In addition, that the LNT101 endolysin had the antibacterial ability in various gram negative bacterium was confirmed by a CFU reduction test, and the result was shown in Table 2 below.
P. aeruginosa
A. baumannii
E. cloacae
K. aerugenes
As shown in Table 2, it was confirmed that it had the bacterium killing effect in a dose dependent manner in all the test gram negative bacterium of 8 kinds of Pseudomonas aeruginosa, 3 kinds of Acinetobacter baumannii, 1 kind of Enterobacter cloacae, and 1 kind of Klebsiella aerogenes, including antibiotic-resistant bacterium.
The Pseudomonas aeruginosa killing ability of the endolysin LNT101 was also confirmed in vivo. As an animal model for the in vivo effectivity evaluation, Galleria mellonella was used. The Galleria mellonella model was divided into the healthy group (non-infection group), Pseudomonas aeruginosa infected group (drug non-administration group), 2 LNT101 administration groups in which LNT101 was administered into the infection model, Colistin administration group in which colistin was administered into the Pseudomonas aeruginosa infected model, and combination administration group in which LNT101 and colistin were administered in combination into the Pseudomonas aeruginosa infected model to progress an experiment, and 10 of Galleria mellonella per each group was used.
The Pseudomonas aeruginosa infected model was prepared by infecting Pseudomonas aeruginosa PA01 at the LD80 concentration, 50 CFU/larva, and LNT101 was administered at 0.6 μg/larva (3 mg/Kg) and 6 μg/larva (30 mg/Kg), respectively. Colistin was administered at 0.5 μg/larva (2.5 mg/Kg), and for administration in combination, Colistin at 0.5 μg/larva (2.5 mg/Kg) and LNT101 at 6 μg/larva (30 mg/Kg) were administered. As the result of observation for 72 hours, the viability of Galleria mellonella was shown in
The synergy effect by treatment in combination of the polymyxin-based antibiotic having a mechanism acting on the cell membrane of bacterium and endolysin LNT101 was confirmed. Specifically, polymyxin B (32 μg/ml-0.03 μg/ml) and colistin (128 μg/ml-0.1 μg/ml) were diluted by ½ per well in a microplate, and then a LNT101 endolysin 1 μM combination treatment group and the same amount of PBS treatment group was made. In all wells, P. aeruginosa 1×105 CFU/ml was treated by total 100 μl. After that, they were cultured at 37° C. for 18 hours. MIC (Minimum Inhibitory Concentration) means the concentration value of the minimum polymyxin-based antibiotic of the well where bacterium did not grow, and the MIC test was performed by broth microdilution technique.
The MIC change of the polymyxin-based antibiotic by combination treatment of the polymyxin-based antibiotic (polymyxin B, Colistin) and LNT101 endolysin at various concentrations was confirmed, and shown in Table 3 below.
As shown in Table 3, it was confirmed that the MIC of the polymyxin-based antibiotics, polymyxin B and Colistin was reduced by 50%, in case of use in combination of the LNT101 endolysin.
The endolysin LNT101 separated and purified in the Example 1.3 (SEQ ID NO: 1) was composed of PG_binding_1 domain (10-65 amino acid) and transglycosylase SLT domain (95-179 amino acid).
Eleven amino acids were substituted for LNT101 endolysin and other amino acid sites with the dominant amino acid sequence of the comparative protein, by comparing the amino acid sequence of endolysin LNT101 PG_binding_1 domain with glycoside hydrolase family 25 of Thermoanaerobacterium phage THSA-485A (accession no. YP_006546280.1), peptidoglycan binding protein of Serratia phage phiMAM1 (accession no. YP_007349105.1), Serratia phage 2050H1 peptidoglycan binding protein (accession no. ASZ78903.1), putative peptidoglycan binding protein of Serratia phage vB_SmaA_3M (accession no. AYP28388.1), putative peptidoglycan binding protein of Enwinia phage vB_EamM-Bue1 (accession no. AV022912.1), putative peptidoglycan binding protein of Pseudomonas phage Noxifer (accession no. YP_009609055.1), endolysin of Salmonella phage Mutine (accession no. AUG88272.1) and peptidoglycan binding protein of Salmonella phage bering (accession no. QIQ61961.1), which have a similar amino acid sequence, by BLASTp analysis.
Four amino acids were substituted for LNT101 endolysin and other amino acid sites with the dominant amino acid sequence of the comparative protein, by comparing the amino acid sequence of LNT101 endolysin transglycosylase SLT domain with tail fiber protein of Pseudomonas phage Noxifer (accession no. YP_009609112.1), hypothetical protein SL2_199 of Pseudomonas phage SL2 (accession no. YP_009619739.1), putative endolysin of Pseudomonas phage KTN4 (accession no. ANM44938.1), PHIKZ144 of Pseudomonas phage phiKZ (accession no. NP_803710.1), tail fiber protein of Pseudomonas phage Psa21 (accession no. QBJ02724.1) and hypothetical protein of Pseudomonas phage vB_PaeM_PS119XW (accession no. QEM41943.1), which have a similar amino acid sequence, by BLASTp analysis.
15 substituted amino acid sequence parts were modified in the LNT101 gene sequence part in consideration of codon usage (SEQ ID NO: 7), and this was named LNT102 (SEQ ID NO: 6). The comparison of the sequences of LNT101 and LNT102 was shown in
The sequences of the described LNT102 and LNT101 were summarized in Table 4 below:
After transforming the prepared pBT7-LNT102 plasmid into the E. coli BL21-Star(DE3) strain (Invitrogen), it was cultured in LB broth (1% Tryptone, 0.5% (w/v) Yeast extract, 0.5% (w/v) NaCl) by OD600=0.5. After that, 1 mM IPTG (Isopropyl β-d-1-thiogalactopyranosid) was added and then cultured with shaking at 37° C. for 4 hours. After cell harvest, it was resuspending with lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole), and 1 mM PMSF and 1 mg/ml lysozyme were added and it was left on ice for 30 minutes. Cells were lysed by sonication, and they were centrifuged at 13,000 rpm for 40 minutes to obtain the supernatant. This was passed through a column in which Ni-NTA agarose resin (Qiagen) was packed. Then, after washing with wash buffer (50 mM NaH2PO4, 300 mM NaCl, 30 mM imidazole), it was eluted with elution buffer (50 mM NaH2PO4, 300 mM NaCl, 300 mM imidazole), to purify LNT102 protein (including 6× His tag).
The purity of the LNT102 protein was confirmed by 15% SDS-PAGE and the concentration of the LNT102 protein was measured by Bradford assay. The result of confirming each reactant obtained in the process of purification by SDS-PAGE was shown in
In the present example, the bacterium killing ability and target bacterium spectrum of the endolysin LNT102 purified in Example 2.2 for various gram negative bacterium were confirmed.
For this, Endolysin LNT101 and LNT102 at a concentration of 2 μM and each 1×106 CFU of Pseudomonas aeruginosa (PA01; ATCC 15692), Acinetobacter baumannii (ATCC 17978), Escherichia coli (ATCC 8739), Klebsiella pneumoniae (ATCC 13883), Enterobacter aerogenes (CCARM 16006), and Salmonella enteritidis (ATCC 13076) was added in reaction buffer (20 mM Tris-Cl, pH 7.5) so that the final volume was 200 μl, and it was left at 37° C. for 2 hours. In 2 hours, the number of colonies of each gram negative bacterium was confirmed, and the result was shown in
The endolysin LNT102 at a concentration of 0.1, 0.5, 2.5 μM, and each 1×106 CFU of Pseudomonas aeruginosa, Acinetobacter baumannii, and Escherichia coli was added in reaction buffer (20 mM Tris-Cl, pH 7.5) so that the final volume was 200 μl, and it was left at 37° C. for 2 hours. In 30 minutes, 1 hour and 2 hours, and the result was shown in
A synergy effect by combination treatment of a polymyxin-based antibiotic having a mechanism acting on the cell membrane of bacterium and endolysin LNT102 purified in Example 2.2 was confirmed. Specifically, after treating 4 μg/ml Polymyxin B to the 96 well microplate row A, serial dilution was performed by ½ in rows B-G. Polymyxin was not treated to the row H. After treating 8 μg/ml Colistin to the 96 well microplate row A, a combination treatment group in which LNT102 endolysin 1 μM was further treated and a PBS treatment group in the same amount were made. All wells were treated with Pseudomonas aeruginosa and Escherichia coli in an amount of 1×105 CFU/ml, total 100 μl, respectively. After that, they were cultured at 37° C. for 18 hours. MIC (Minimum Inhibitory Concentration) means a concentration value of a minimum polymyxin-based antibiotic of a well in which bacterium do not grow, and the MIC test was performed by broth microdilution technique according to the standard test method of CLSI (Clinical and Laboratory Standards Institute) (Clinical and Laboratory Standards Institute. Methods for dilution antimicrobial susceptibility tests for bacterium that grow aerobically; approved standard. 11th ed. Document M07. Wayne, PA: CLSI; 2018).
The MIC change of the polymyxin-based antibiotics by combination treatment of the polymyxin-base antibiotics (polymyxin B, Colistin) at various concentrations and LNT102 endolysin was measured and shown in Table 5 below.
P. aeruginosa
A. baumannii
E. coli(ATCC
As shown in Table 5, it was confirmed that the MIC of the polymyxin-based antibiotics, polymyxin B and Colistin was reduced by 1/16 times at maximum, in case of use in combination of the LNT102 endolysin. This result shows that a significantly increased antibiotic effect can be obtained when a conventional antibiotic, for example, a polymyxin-based antibiotic and LNT102 endolysin are treated in combination.
Endolysin LNT101 (SEQ ID NO: 1) is composed of PG_binding_1 domain (10-65 amino acid) and transglycosylase SLT domain(95-179 amino acid). The endolysin LNT101 variant of SEQ ID NO: 6 (hereinafter, named endolysin LNT102) was prepared by adding 15 amino acid mutations in the endolysin LNT101 (SEQ ID NO: 1).
The coding gene of the endolysin LNT102 (SEQ ID NO: 7) was inserted to pBT7 plasmid to prepare pBT7-LNT102 plasmid for endolysin LNT102 expression. The prepared pBT7-LNT102 plasmid was transformed into E. coli BL21-Star(DE3) strain (Invitrogen), it was cultured in LB broth (1% Tryptone, 0.5% (w/v) Yeast extract, 0.5% (w/v) NaCl) by OD600=0.5. After that, 1 mM IPTG (Isopropyl β-d-1-thiogalactopyranosid) was added, and then it was cultured with shaking at 37° C. for 4 hours. After cell harvest, it was resuspended with lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole), and 1 mM PMSF and 1 mg/ml lysozyme were added and it was left on ice for 30 minutes. Cells were lysed by sonication, and it was centrifuged at 13,000 rpm for 40 minutes to obtain the supernatant. This was passed through a column in which Ni-NTA agarose resin (Qiagen) was packed. Then, after washing with wash buffer (50 mM NaH2PO4, 300 mM NaCl, 30 mM imidazole), it was eluted with elution buffer (50 mM NaH2PO4, 300 mM NaCl, 300 mM imidazole), to purify the LNT102 protein (including 6Δ His tag).
The purity of the LNT102 protein was confirmed by 15% SDS-PAGE, and the concentration of the LNT102 protein was measured by Bradford assay. As the result of confirming each reactant obtained during the process of purification by SDS-PAGE, the molecular weight of the purified LNT102 protein was about 31 kDa.
A fusion polypeptide in which Cecropin A was fused in the prepared LNT102 protein was prepared. Specifically, a polynucleotide (SEQ ID NO: 11) encoding a fusion polypeptide (named LNT103) (SEQ ID NO: 10) comprising [Cecropin A (SEQ ID NO: 8)]-[linker (GSGSGS) (SEQ ID NO: 12)]-[endolysin (named LNT102) (SEQ ID NO: 6)] from the N-terminus in order was inserted into pET 21a (Novagen) plasmid to prepare plasmid pET-LNT103 for LNT103 expression. After transforming the prepared pET-LNT103 plasmid into E. coli BL21-Star(DE3) strain (Invitrogen), it was cultured in LB broth (1% (w/v) Tryptone, 0.5% (w/v) Yeast extract, 0.5% (w/v) NaCl) by OD600=0.5.
After that, after adding 1 mM IPTG (isopropyl β-D-1-thiogalactopyranoside), it was cultured with shaking at 25° C. for 6 hours. After cell harvest, it was resuspended with lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole), and 1 mM PMSF (phenylmethylsulfonyl fluoride) and 1 mg/ml lysozyme were added, and it was left on ice for 30 minutes. Cells were lysed by sonication, and it was centrifuged at 13,000 rpm for 40 minutes to obtain the supernatant. The obtained supernatant was passed through a column in which Ni-NTA agarose resin (Qiagen) was packed. Then, after washing with wash buffer (50 mM NaH2PO4, 300 mM NaCl, 30 mM imidazole), it was eluted with elution buffer (50 mM NaH2PO4, 300 mM NaCl, 300 mM imidazole). It was purified/confirmed by 15% SDS-PAGE and the concentration was measured by Bradford assay.
The obtained SDS-PAGE result was shown in
In addition, by the same method as the above method, a fusion polypeptide in which Cecropin A was fused to endolysin LNT101 (SEQ ID NO: 1) (CecA-LNT101; SEQ ID NO: 13) was produced. For the produced CecA-LNT101, SDS-PAGE was performed by the aforementioned method, to confirm that the molecular weight of CecA-LNT101 was 34 kDa.
On the other hand, for convenience of the test, the first amino acid M of the purified fusion polypeptide LNT103 (SEQ ID NO: 10) and CecA-LNT101(SEQ ID NO: 13) was substituted with MAS (Met-Ala-Ser), and it was produced in a form in which an extra sequence and a His tag were added to the C-terminus (SEQ ID NO: 15 or SEQ ID NO: 16), and in the following test, SEQ ID NO: 15 was used as LNT103, and SEQ ID NO: 16 was used as CecA-LNT101, respectively.
The amino acid sequences and nucleic acid sequences of the polypeptide and their coding genes described in the present example were summarized in Table 6 below:
MASKWKLFKKIEKVGQNIRDGIIKAGPAVAVVGQATQIAKGSGSGSMGTVLKRGD
MASKWKLFKKIEKVGQNIRDGIIKAGPAVAVVGQATQIAKGSGSGSMGTVLKRGD
In order to evaluate the outer membrane permeabilization activity of the gram negative bacterium of the fusion polypeptide prepared in the Example 3.2, NPN uptake assay was performed. As representative gram negative bacterium, Pseudomonas aeruginosa and Acinetobacter baumannii were used for the test.
Pseudomonas aeruginosa (PA01; ATCC 15692) or Acinetobacter baumannii (ATCC 19606) was cultured by OD600=0.3 and centrifuged at 1,000 g for 10 minutes, and then it was resuspended with a ½ volume of 5 mM HEPES (Hydroxyethyl piperazine Ethane Sulfonic acid) (pH 7.2). 40 μM NPN (1-N-phenylnaphthylamine) solution (40 μM NPN in 5 mM HEPES, pH7.2) 50 μl, the test substance (LNT101, LNT102 or LNT103) 50 μl, and strain suspension solution 100 μl (total 200 μl) were added to a microplate and reacted at 37° C. for 1 hour. Then, fluorescence was measured under the condition of excitation 350 nm and emission 420 nm with a microplate reader (Infinite M200 Pro, TECAN). As a positive control group, Cecropin A (SEQ ID NO: 8), EDTA, or polymyxin B was used, and as a negative control group, 10 μM NPN solution was used. All the experiments were performed in 3 sets.
The obtained result was shown in
On the other hand, in order to confirm the difference in the outer membrane permeabilization activity against gram negative bacterium (Pseudomonas aeruginosa (PA01; ATCC 15692) and Acinetobacter baumannii (ATCC 19606)) according to the concentration of LNT103, LNT103 was treated in an amount of 0.29 μM, 0.88 μM, or (2.65 μM), and 2 μM of cecropin A and polymyxin B, and 1 mM of EDTA were treated. The obtained result was shown in C and D of
In order to confirm the killing ability against gram negative bacterium of the fusion polypeptide LNT103 prepared in Example 3.2, CFU reduction evaluation was performed for various gram negative bacterium. In order to perform the CFU reduction evaluation, 2 μM of each of LNT101, LNT102, and LNT103 prepared in Example 3, and 1×106 CFU of each of Pseudomonas aeruginosa (PA01; ATCC 15692), Acinetobacter baumannii (ATCC 17978), Escherichia coli (ATCC 8739), Klebsiella pneumoniae (ATCC 13883), and Enterobacter aerogenes (CCARM 16006) were added to reaction buffer (20 mM Tris-Cl, pH7.5) so that the final volume was 200 μl, and it was left at 37° C. for 2 hours. After that, the number of the colonies of each gram negative bacterium was confirmed to compare and evaluate the antibiotic effect of each polypeptide. As a control group, the PBS treatment group (treating PBS in the same volume as the fusion polypeptide) was used.
The obtained result was shown in
In addition, the same test was performed by using the fusion polypeptide LNT103, and CecA-LNT101 at a concentration of 2 μM, and the result was shown in
On the other hand, in order to compare the antibacterial activity in case that a protein other than endolysin (LNT102 or LNT101) was fused to Cecropin A, the killing ability against gram negative bacterium of the fusion polypeptide LNT103 and cecropin A-EGFP fusion protein (a protein in which EGFP (GenBank Accession No. AAB02572.1) having no endolysin activity by replacing LNT102 in LNT103) was evaluated. For this, the proteins were treated in an amount of 0.2 μM or 2 μM, respectively, to perform the CFU reduction test. The obtained result was shown in
Furthermore, the killing ability against gram negative bacterium according to the concentration and/or treatment time of the fusion polypeptide LNT103 was tested. Specifically, the fusion polypeptide LNT103 at a concentration of 0.1, 0.3, 0.9, or 2.7 μM and 1×106 CFU of Pseudomonas aeruginosa (PA01; ATCC 15692) or Acinetobacter baumannii (ATCC 19606) were added to reaction buffer (20 mM Tris-Cl, pH7.5) so that the final volume was 200 μl, and it was left at 37° C. for 2 hours, and then the number of the colonies was confirmed, and the result was shown in A of
In addition, the fusion polypeptide LNT103 at a concentration of 2 μM and 1×106 CFU of Pseudomonas aeruginosa (PA01; ATCC 15692) were added to reaction buffer (20 mM pH7.5) so that the final volume was 200 μl, and in 0, 20 minutes, 40 minutes and 60 minutes at 37° C., the number of the colonies was confirmed, and the result was shown in B of
Moreover, the fusion polypeptide LNT103 at a concentration of 1 μM and 1×106 CFU of Acinetobacter baumannii (ATCC 19606) were added to reaction buffer (20 mM pH7.5) so that the final volume was 200 μl, and in 0, 1 minute, 3 minutes, 5 minutes and 10 minutes at 37° C., the number of the colonies was confirmed, and the result was shown in C of
As shown in A, B and C of
In addition, MIC (Minimal Inhibitory Concentration) and MBC (Minimal Bactericidal Concentration) of the fusion polypeptide LNT103 were measured. The MIC and MBC were performed by broth microdilution technique according to the standard test method of CLSI (Clinical and Laboratory Standards Institute), and in the corresponding test method, it was performed by using CAA media (Casamino acid 5 g/L, K2HPO4 5.2 mM, MgSO4 1 mM) instead of MH broth (Casein acid hydrolysate 17.5 g/L, Beef extract 3.0 g/L, Starch 1.5 g/L. pH 7.3) (Clinical and Laboratory Standards Institute. Methods for dilution antimicrobial susceptibility tests for bacterium that grow aerobically; approved standard. 11th ed. Document M07. Wayne, PA: CLSI; 2018). Specifically, the LNT103 was treated at a concentration of 64 μg/ml to a 96 well microplate row A, and the LNT103 was treated at a concentration where serial dilution was performed by ½ in rows B-G. To the row H, LNT103 was not treated. After that, the corresponding bacterium strain (type or QC, CCARM and clinical separation strain; See Tables 2-4) was treated in an amount of 5×105 CFU/ml, total 100 μl in all wells. Then, it was cultured at 37° C. for 18 hours. MIC was determined to be the lowest concentration at which no colonies were identified by confirming the number of the colonies in each well. The obtained result was shown in Table 7 to Table 9 below:
A. baumannii
P.
aeruginosa
E. coli
The synergy effect of killing ability against gram negative bacterium by combination treatment of a polymyxin-based antibiotic having a mechanism acting on the cell membrane of bacterium and the fusion polypeptide LNT103 was confirmed. Specifically, colistin (polymyxin E) of 16 μl/ml was treated to a 96 well microplate column 1, and it was treated at a concentration where serial dilution was performed by ½ in columns 2-11. The LNT103 of 16 μg/ml was treated to row A, and it was treated at a concentration where serial dilution was performed by ½ in rows B-G. After that, Acinetobacter baumannii (ATCC 19606) was treated in an amount of 5×105 CFU/ml, total 100 μl in all wells. Then, it was cultured at 37° C. for 18 hours, and the FIC (Fractional Inhibitory Concentration) index value was calculated by the following equation:
FIC index=FICA+FICB=(CA/MICA)+(CB/MICB)
(CA and CB are each concentration of substances treated in combination (polymyxin E and LNT103), and MICA and MICB are MIC of single drug,
FIC value: synergy effect <0.5; antagonism >4; additive 0.5-4)
As the result of the test, the FIC index value was shown as 0.375, and it was confirmed that there was a synergy effect between the two substances.
In vitro toxicity of the fusion polypeptide LNT103 was evaluated by cell cytotoxicity assay (WST assay) using Huh-7 cell line and hemolysis assay using sheep blood (MB cell).
The process of progressing the cell cytotoxicity assay was as follows. The Huh07 cell culture solution was prepared and aliquoted in an amount of 1×109 cells/well per well in a 96 well plate, and it was cultured in a CO2 incubator for 24 hours. Then, PBS as a negative control and 1% (w/v) triton X-100 as a positive control were treated, and as an experimental group, the fusion polypeptide LNT103 was treated at a concentration of 0.25, 0.5, or 1 mg/ml, and then it was left in a CO2 incubator for 24 hours and 48 hours. Then, the cell viability was measured using D-Plus™ CCK cell viability assay kit (Dongin LS), and the result was shown in
The process of progressing the hemolysis assay was as follows. After mixing 3 ml sheep blood and 14 ml PBS (pH 7.2), it was centrifuged at 1000 g, 4° C. for 5 minutes to remove the supernatant. After that, PBS was filled again as much as the removed volume and the above washing was progressed 4 times in total. Hemoglobin lysed through washing was removed, and after the last centrifugation, 400 μl of sunken RBC (red blood cell) was dissolved in 9.6 ml PBS to make 4% (v/v) blood solution. After mixing 50 μl of the sample and 50 μl of 4% (v/v) blood solution in a 96 well microplate, it was reacted at 37° C. for 1 hour. For an experimental group, the fusion polypeptide LNT103 was treated from 128 μg/ml to 2 μg/ml in a 2-fold dilution range (PBS, pH 7.2), and for a positive control group, 0.1% (w/v) Triton X-100 and for a negative control group, PBS were treated. After reacting for 1 hour, the microplate was centrifuged at 1000 g, 4° C. for 5 minutes, and 50 μl of the supernatant was collected and it was transferred to a new microplate and the absorbance was measured at 570 nm (Infinite M200 Pro, TECAN). The obtained result was shown in
For in vivo effectivity evaluation of the fusion polypeptide LNT103, the viability was confirmed in an Acinetobacter baumannii ATCC 19606 systemic infection mouse model was confirmed.
The process of progressing an animal experiment was as follows. Mice were used when ICR male 4-week-old mice were purchased and a one-week adaptation period was passed, and the weight was 20˜21 g at 5-6 weeks of age. The Acinetobacter bacteria were mixed with 10% mucin at 1:1, and 0.5 ml of 2×108 CFU/ml (5% mucin) of the bacterial solution was inoculated intraperitoneally into mice. In 1 hour and 4 hours after infection, LNT103 20 mpk and LNT103 100 mpk, and colistin 20 mpk as a comparative group were administered, respectively, through a subcutaneous injection. After that, the viability for 96 hours was confirmed, and the result was shown in
From the above description, those skilled in the art to which the present application pertains will be able to understand that the present application may be executed in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that Examples described above are illustrative and not restrictive in all respects. The scope of the present application should be construed as that all changed or modified forms derived from the meaning and scope of claims to be described below and equivalent concepts rather than the detailed description are included in the scope of the present application.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2020-0061906 | May 2020 | KR | national |
| 10-2020-0108498 | Aug 2020 | KR | national |
| 10-2021-0019108 | Feb 2021 | KR | national |
This invention was made with government support under Ref No: 1415184431 awarded by the Korea Evaluation Institute of Industrial Technology, Ministry of Trade, Industry and Energy.
| Number | Date | Country |
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| 2013-541333 | Nov 2013 | JP |
| 2020-500935 | Jan 2020 | JP |
| 10-2012-0095345 | Aug 2012 | KR |
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| Number | Date | Country | |
|---|---|---|---|
| 20220348895 A1 | Nov 2022 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/KR2021/006302 | May 2021 | US |
| Child | 17853027 | US |
